Abstract Hemophilia B is an X-linked recessive monogenic disorder affecting approximately 1 in 25,000 males, characterized by mutations which result in non functional or aberrant Factor IX (FIX) coagulation activity. These mutations can lead to a phenotype of spontaneous bleeding into joints and soft tissue, depending on the severity of the disease. Current clinical treatment of hemophilia B consists of life-long therapy with injections of FIX. The cost of treatment is expensive as well as physically invasive as patients must undergo recurrent intravenous injections. Thus, one of the core objectives of gene therapy for Hemophilia B is the establishment of long term therapeutic gene expression of coagulation factor IX in preclinical models of coagulation deficiency. Recent advancements in gene editing technology has given rise to the possibility of targeting specific loci for stable integration and long term expression of therapeutic transgenes. Herein, we hypothesize adenoviral-mediated gene therapy can establish long term therapeutic FIX expression following homology directed repair using a CRISPR/Cas9-based system targeted to the ?safe harbor? ROSA26 locus in murine knockout and missense models of Hemophilia B. Human adenovirus C serotype 5 is a well characterized viral vector that is ideally suited for this proof of concept project due to its large cloning capacity in replication deficient vectors, high titers, ability to infect both dividing and non dividing cells, as well as its native tropism to liver tissue, the natural site of FIX production. In addition, adenovirus has been utilized as a successful delivery platform for CRISPR/Cas9 strategies and has been shown to be an excellent vector for donor DNA. FIX integration at the ROSA26 locus will be achieved through co-injection of a vector containing mFIX, flanked by ROSA26 specific sequences for homologous recombination, and a virus encoding a ROSA26 specific guideRNA and the Cas9 endonuclease. The dual vector system will first be validated in vitro using murine hepatocytes then utilized for gene delivery in vivo using the well characterized FIX KO mouse strain, which faithfully recapitulates the human disease. The mice will be analyzed for levels of FIX expression, therapeutic FIX activity, efficacy of the system compared to other strategies, and improved coagulation phenotypes while monitoring for vector mediated toxicity and inhibitor antibody formation. If successful this study will represent the feasibleness of CRISPR/Cas9 system for the targeted integration of therapeutic genes in post embryonic somatic tissue while providing further information on the utility of CRISPR/Cas9 system.